Shuttle vectors developed from Streptococcus thermophilus native plasmid

Abstract

pER8 (2.2 kb), a native plasmid of Streptococcus thermophilus ST108, was used to develop pMEU-series shuttle vectors. In addition to the replication function of the pER8, these vectors contain origin of replication and beta-lactamase gene (bla) of Escherichia coli vector pUC18/19, the cat gene of pC194, and the pPV141-borne erm determinant of Staphylococcus hyicus ssp. chromogenes 3688. pMEU5a, pMEU5b, pMEU6a, and pMEU6b (all 5.7 kb in size) contain bla and erm markers, are capable of transforming E. coli and S. thermophilus at frequencies in the order of 10(5)-10(6) and 10(3) colony forming units (CFU)/microgram DNA, respectively, and are highly stable in the two host systems. pMEU9 and pMEU10 (both 6.9 kb) contain the cat marker in addition to the DNA elements found in pMEU5-6. These plasmids are also highly effective in transforming E. coli (at ca. 6 x 10(5) CFU/microgram DNA) and S. thermophilus (ca. 10(3) CFU/microgram DNA). Although expression of the resistance markers is not completely consistent, pMEU9 and pMEU10 remain important shuttle vectors for clonal selection by an insertional inactivation method.